RU2798270C2 - Method for obtaining bioethanol from lignocellulose raw material - Google Patents

Abstract

FIELD: alcohol industry.

SUBSTANCE: method for producing bioethanol from lignocellulosic raw materials provides for delignification of raw materials at atmospheric pressure, combining the stages of enzymatic hydrolysis and alcoholic fermentation, and isolation of bioethanol from mash. At the same time, miscanthus is used as a raw material, the delignification of raw materials with a moisture content of 36% is carried out at atmospheric pressure with dilute acetic acid with a concentration of 10% at 94-96°C with the addition of a 30% hydrogen peroxide solution, distilled water and concentrated sulfuric acid. Enzymatic hydrolysis is carried out at pH 5.0 at a temperature of 45°C for 5 hours with the Cellulade Ultra enzyme. Fermentation is carried out using a consortium of seed yeast Pichia stipites Y7124, Candida shehatae NCL3501, Kluyveromyces marxianus Y-4290 and Zymomonas mobilis 113, taken in a ratio of 1:1:1:1.

EFFECT: invention provides the yield of bioethanol from 1 ton of raw materials equal to 25.5 dal.

1 cl, 3 ex

Description

The invention relates to methods for producing bioethanol from vegetable raw materials.

Increasing global energy demand, unsustainable and expensive oil resources, and concerns about global climate change have prompted the development of renewable energy sources that can supplement fossil fuels. In this regard, lignocellulosic biomass has a high potential for the production of biofuels (eg bioethanol) and other valuable substances based on the concept of biorefining.

Biomass of various origins serves as an industrial source for obtaining modern bioethanol. The traditional raw materials for the production of bioethanol are sugar-containing raw materials (sugar cane, sugar beets), as well as starch-containing raw materials (potatoes and cereals). However, these types of raw materials are edible, their use is not only expensive, but can also worsen food security. Second generation bioethanol is produced from non-food feedstocks: wood, seaweed, cellulose-containing non-wood feedstocks such as agricultural waste and energy plant biomass.

It is known that native cellulose-containing raw materials are quite strong, since the plant matrix consists of several polymers: cellulose, hemicellulose, and lignin, which together form a composite material that is more resistant to physical and chemical factors than individual components. In order to split individual polymers, it is necessary to destroy the composite matrix, for which the native raw material must be subjected to chemical or physicochemical treatment. After the destruction of the composite matrix, it is necessary to degrade the polymers to monomers: cellulose to glucose, hemicellulose to monomeric pentoses and hexoses. This degradation can be carried out by chemical or enzymatic hydrolysis.

Enzymatic hydrolysis of lignocellulose is carried out under mild conditions, which, unlike chemical hydrolysis, excludes the formation of toxic intermediates, and also guarantees a high yield of fermentable sugars.

The resulting solution of monomers is subjected to alcoholic fermentation and, using the built-in enzyme systems of microorganisms (yeast or bacteria), is converted into a mash - a suspension consisting of water, bioethanol, side and secondary substances formed during fermentation, solid particles of unreacted lignocellulosic substrate and cells of microorganisms. Bioethanol is isolated from the mash by membrane methods or by rectification.

The study of the prior art revealed essentially similar known technical solutions, for example, a method for producing bioethanol from lignocellulosic material according to US patent No. 7189306, which includes pre-treatment, enzymatic hydrolysis, alcoholic fermentation of enzymatic hydrolysates and isolation of bioethanol from mash.

The disadvantages of the described method include the complexity of preparing lignocellulosic raw materials for the production of bioethanol, namely the need to use an energy-intensive steam explosion to create high pressure and use expensive equipment in the process - a distillation column of a special design.

The closest is the method for obtaining bioethanol from lignocellulosic raw materials according to the RF patent No. 2581799, including pre-treatment of raw materials, enzymatic hydrolysis, alcoholic fermentation of enzymatic hydrolysates, isolation of bioethanol from mash.

The disadvantages of the described method include the need to use special mechanical equipment in the pre-treatment of raw materials for enzymatic hydrolysis. As a result of mechanical processing, the raw material matrix is destroyed under the influence of gravity, including the shear and tear forces that occur between the material and the drum, as well as the forces created by the collision of the falling material and the bottom of the drum.

The objective of the proposed technical solution is to create an effective method for producing bioethanol from lignocellulosic rapidly renewable raw materials, which allows for the industrial scaling of the process using standard equipment.

The problem is solved by the proposed method for obtaining bioethanol from lignocellulosic raw materials, which includes delignification of raw materials at atmospheric pressure, enzymatic hydrolysis, alcoholic fermentation, isolation of bioethanol from mash, while the delignification of raw materials with a moisture content of 36% is carried out at atmospheric pressure with dilute acetic acid with a concentration of 10% at 94-96°C with the addition of a 30% hydrogen peroxide solution, distilled water and concentrated sulfuric acid. Enzymatic hydrolysis is carried out at pH 5.0 at a temperature of 45°C for 5 hours As raw materials used rapidly renewable lignocellulosic raw materials: miscanthus.

The proposed technical solution differs from the closest analogue in that the delignification of raw materials with a moisture content of 36% is carried out at atmospheric pressure with dilute acetic acid with a concentration of 10% at 94-96°C with the addition of a 30% hydrogen peroxide solution, distilled water and concentrated sulfuric acid. Enzymatic hydrolysis is carried out at pH 5.0 at a temperature of 45°C for 5 hours, and as a raw material is used rapidly renewable lignocellulosic raw material: miscanthus.

The purpose of delignification is to change the physical features and chemical composition/structure of the hydrolysable part of the raw material, which makes the cellulose more accessible for enzymatic hydrolysis and its conversion into a sugar solution. In the proposed technical solution, chemical processing is carried out at atmospheric pressure, which allows using simple standard capacitive equipment when scaling the process, as well as creating safer working conditions than in the case of using raw materials processing modes under pressure.

For raw materials with a moisture content of 36% (miscanthus), it is proposed to use delignification in the following sequence: at atmospheric pressure, adding dilute acetic acid with a concentration of 10% at 94-96 ° C with the addition of a 30% hydrogen peroxide solution, distilled water and concentrated sulfuric acid. The main function of the action of the acid is the hydrolysis of hemicelluloses and the destruction of the lignin structure in such a way that the availability of the cellulose fraction for enzymes in the delignified raw material increases, this is effective for wet raw materials.

The purpose of enzymatic hydrolysis is the degradation of polymers to monomers. Cellulose is broken down to glucose, hemicellulose to monomeric pentoses and hexoses. For enzymatic hydrolysis, available commercial enzyme preparations with cellulase activity are used. Enzymatic hydrolysis is carried out by the Cellulade Ultra enzyme under mild conditions: active acidity 5.0 units. pH, at a temperature of 45°C for 5 hours, which eliminates the formation of toxic intermediates, and also guarantees a high yield of fermentable sugars. With the indicated active acidity, the highest yields of reducing substances are achieved, with a deviation up or down from the indicated level of active acidity, the yield of reducing substances is significantly reduced (by 30% or more), which is associated with conformational changes in enzyme molecules and a decrease in enzymatic activity.

At a temperature of 45°C, the maximum yields of reducing substances were recorded. A decrease in the hydrolysis temperature below 35°C leads to a loss in the yield of sugars by 10%, which can be explained by a decrease in the rate of the chemical reaction with decreasing temperature according to the van't Hoff rule. An increase in temperature above 65°C is critical due to the thermal denaturation of the enzyme, which is explained by the loss of the quaternary or tertiary structure of the protein upon heating, and leads to a decrease in the yield of reducing substances by 50%.

The purpose of the alcoholic fermentation stage is the conversion of the reducing substances of the environment into bioethanol using enzyme systems of a consortium of microorganisms of various genera and species. In the proposed technical solution, alcoholic fermentation is carried out using microorganisms Pichia stipites Y7124, Candida shehatae NCL3501, Kluyveromyces marxianus Y-4290 and Zymomonas mobilis 113, the ratio of microorganisms is 1:1:1:1.

Carrying out the simultaneous process of enzymatic hydrolysis and alcoholic fermentation makes it possible to reduce the duration of the stages by 2.5 times and eliminate the filtration of the intermediate product - the enzymatic hydrolyzate. This will reduce the cost of obtaining bioethanol and simplify the technological process, which is important for its successful scaling.

As a rapidly renewable lignocellulosic raw material, the proposed method uses miscanthus, which is an industrial crop, for which there is no need to allocate plantations of fertile arable land. After 15 years, the vegetation of the plantation stops and a new one is laid. The calculation was made based on the minimum productivity of miscanthus in Western Siberia (10 t/ha/year). Thus, over 15 years, the productivity of the miscanthus plantation will be 185 tons per hectare, while the accumulation of hardwood biomass over the same period is 54-68 tons per hectare. The results of determining the chemical composition confirm the content of lignin 20%, hemicellulose 20%.

It has been established by gas-liquid chromatography that the enzymatic method of cellulose hydrolysis of lignocellulosic raw materials makes it possible to obtain bioethanol with a low content of esters and fusel oils. There is no methanol in bioethanol.

In the proposed technical solution, the stage of isolation of bioethanol can be carried out by any known method using standard equipment.

To explain the described technical solution, examples of the proposed method are given below.

Example 1

Miscanthus with a moisture content of 36% is delignified at atmospheric pressure with dilute acetic acid at a concentration of 10% (30 ml) at 94-96 ° C with the addition of a 30% hydrogen peroxide solution (20 ml), distilled water (50 ml) and concentrated sulfuric acids (2 ml). The obtained colorless cellulose is separated by centrifugation at 3900±50 rpm and washed with two portions of distilled water.

Enzymatic hydrolysis is carried out in a fermenter in an aqueous medium. Hydrolysis temperature 45°C, active acidity is set at 5.0 units. pH. The substrate concentration is 80 g/l. The enzyme is applied as follows: "Cellulade Ultra" at the rate of 30 mg of enzyme per 1 liter of substrate. The duration of enzymatic hydrolysis is 5 hours. Then a consortium of seed yeast (Pichia stipites Y7124, Candida shehatae NCL3501, Kluyveromyces marxianus Y-4290 and Zymomonas mobilis 113, the ratio of microorganisms is 1:1:1:1) is added and alcohol fermentation is carried out for the next three days, combined with saccharification. Bioethanol is isolated and purified by rectification. The yield of bioethanol from 1 ton of miscanthus is 25.5 dal.

Example 2

Miscanthus with a moisture content of 36% is delignified at atmospheric pressure with dilute acetic acid at a concentration of 10% (30 ml) at 94-96 ° C with the addition of a 30% hydrogen peroxide solution (20 ml), distilled water (50 ml) and concentrated sulfuric acids (2 ml). The obtained colorless cellulose is separated by centrifugation at 3900±50 rpm and washed with two portions of distilled water.

Enzymatic hydrolysis is carried out in a fermenter in an aqueous medium. Hydrolysis temperature 65°C, active acidity is set at 5.0 units. pH. The substrate concentration is 80 g/l. The enzyme is applied as follows: "Cellulade Ultra" at the rate of 20 mg of enzyme per 1 liter of substrate. The duration of enzymatic hydrolysis is 10 hours. Next, a consortium of seed yeast (Pichia stipites Y7124, Candida shehatae NCL3501, Kluyveromyces marxianus Y-4290 and Zymomonas mobilis 113, the ratio of microorganisms is 1:1:1:1) is added and alcohol fermentation is carried out for the next three days, combined with saccharification. Bioethanol is isolated and purified by rectification. The yield of bioethanol from 1 ton of miscanthus is 15.3 dal.

Example 3

Miscanthus with a moisture content of 36% is delignified at atmospheric pressure with dilute acetic acid at a concentration of 10% (30 ml) at 94-96 ° C with the addition of a 30% hydrogen peroxide solution (20 ml), distilled water (50 ml) and concentrated sulfuric acids (2 ml). The obtained colorless cellulose is separated by centrifugation at 3900±50 rpm and washed with two portions of distilled water.

Enzymatic hydrolysis is carried out in a fermenter in an aqueous medium. Hydrolysis temperature 35°C, active acidity is set at 5.0 units. pH. The substrate concentration is 80 g/l. The enzyme is applied as follows: "Cellulade Ultra" at the rate of 40 mg of enzyme per 1 g of substrate. The duration of enzymatic hydrolysis is 3 hours. Next, a consortium of seed yeast (Pichia stipites Y7124, Candida shehatae NCL3501, Kluyveromyces marxianus Y-4290 and Zymomonas mobilis 113, the ratio of microorganisms is 1:1:1:1) is added and alcohol fermentation is carried out for the next three days, combined with saccharification. Bioethanol is isolated and purified by rectification. The yield of bioethanol from 1 ton of miscanthus is 10.6 dal.

Thus, the proposed method is efficient and technologically feasible, is implemented on standard equipment and makes it possible to obtain bioethanol from lignocellulosic raw materials, which are characterized by high yields and economic potential of cultivation. The implementation of the method will satisfy the long-existing need for solving the problem with obtaining a technical result - increasing the yield of bioethanol from lignocellulosic raw materials (25.5 dal against 8.5-20.5 dal according to the closest analogue).

Claims (1)

A method for producing bioethanol from lignocellulosic raw materials, including delignification of raw materials at atmospheric pressure, combining the stages of enzymatic hydrolysis and alcoholic fermentation, isolation of bioethanol from mash, characterized in that miscanthus is used as lignocellulosic raw materials, delignification of raw materials with a moisture content of 36% is carried out at atmospheric pressure with diluted acetic acid with a concentration of 10% at 94-96°C with the addition of a 30% hydrogen peroxide solution, distilled water and concentrated sulfuric acid, enzymatic hydrolysis is carried out at pH 5.0 at a temperature of 45°C for 5 hours with the introduction of Cellulade Ultra » in the calculation of 30 mg of enzyme per 1 liter of substrate, then a consortium of seed yeast Pichia stipites Y7124, Candida shehatae NCL3501, Kluyveromyces marxianus Y-4290 and Zymomonas mobilis 113, taken in a ratio of 1:1:1:1, is added, and for three subsequent days, alcoholic fermentation is carried out, combined with saccharification.